Multivesicular body (MVB) sorting is a critical process within the endocytic pathway wherein portions of the endosomal membrane bud into the endosomal lumen. Failure to target activated growth factor receptors into the MVB pathway results in prolonged signaling that can contribute to tumorigenesis and defects in organism development. Defects in MVB targeting of the epithelial sodium channel result in an inherited form of hypertension (Liddle's syndrome). Neurodegenerative diseases have been linked to dysfunction of the MVB pathway, and aberrant trafficking of lipids through this pathway also contributes to a number of human disease states, including atherosclerosis. MVB sorting is mediated by the endosomal sorting complexes required for transport (ESCRT-0, I, II, and III) and associated factors including the AAA-ATPase Vps4. In addition to roles in MVB sorting, the ESCRTs and Vps4 are usurped by enveloped viruses (e.g. HIV-1 and Ebola) to execute their cellular egress. Furthermore, the ESCRTs and Vps4 contribute to membrane abscission during cytokinesis. These examples highlight numerous processes impacted by ESCRT function as well as maladies that arise upon dysfunction. Vps4 and ESCRT-III act in a coordinated manner during membrane deformations of similar topology (away from the cytoplasm) that occur in MVB sorting, viral budding, and membrane abscission. Dissociation of ESCRT-III through Vps4 ATP hydrolysis is required for these processes, while ESCRT-III also regulates Vps4. The studies presented in this proposal apply biochemical approaches to probe the enzymatic activity of Vps4, the regulation of Vps4 by ESCRT-III, and the mechanism by which Vps4-stimulated ESCRT-III dissociation facilitates intralumenal vesicle formation. These inquiries are complemented by in vivo functional analyses in the model organism Saccharomyces cerevisiae. Completion of this experimental plan will provide insight into the conserved evolutionary function of ESCRT-III and Vps4 in membrane constriction during MVB sorting, viral budding and cytokinesis. PUBLIC HEALTH RELEVANCE: Multivesicular body sorting machinery deforms the membrane in a unique manner to permit growth factor receptor downregulation, cellular division and viral budding. Understanding how this process takes place will lead to insights into the prevention of cancer, neurodegenerative diseases and hypertension as well as mechanisms of viral reproduction.